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Cholesterol oxidases are bifunctional flavoenzymes that catalyze the oxida-tion of steroid substrates which have a hydroxyl group at the 3bposition of the steroid ring system. The enzyme is found, in a wide range of bacte-rial species, in two forms: one with the FAD cofactor bound noncovalently to the enzyme; and one with the cofactor linked covalently to the protein. | MINIREVIEW Cholesterol oxidase biochemistry and structural features Alice Vrielink1 and Sandro Ghisla2 3 1 Schoolof Biomedical Biomolecular and ChemicalSciences University of Western Australia Crawley Australia 2 Department of Biotechnology and Molecular Sciences University of Insubria Varese Italy 3 Fachbereich Biologie University of Konstanz Konstanz Germany Keywords cholesterol oxidase enzyme kinetics enzyme mechanism flavoenzyme oxygen channel protein structure redox catalysis Correspondence A. Vrielink School of Biomedical Biomolecular and Chemical Sciences University of Western Australia 35 Stirling Highway - Crawley WA 6009 Australia Fax 61 8 6488 1148 Tel 61 8 6488 3162 E-mail alice.vrielink@uwa.edu.au Received 27 July 2009 revised 7 September 2009 accepted 14 September 2009 Cholesterol oxidases are bifunctional flavoenzymes that catalyze the oxidation of steroid substrates which have a hydroxyl group at the 3 b position of the steroid ring system. The enzyme is found in a wide range of bacterial species in two forms one with the FAD cofactor bound noncovalently to the enzyme and one with the cofactor linked covalently to the protein. Here we discuss compare and contrast the salient biochemical properties of the two forms of the enzyme. Specifically the structural features are discussed that affect the redox potentials of the flavin cofactor the chemical mechanism of substrate dehydrogenation by active-center amino acid residues the kinetic parameters of both types of enzymes and the reactivity of reduced enzymes with molecular dioxygen. The presence of a molecular tunnel that is proposed to serve in the access of dioxygen to the active site and mechanisms of its control by a gate formed by amino acid residues are highlighted. doi 10.1111 j.1742-4658.2009.07377.x Introduction Cholesterol oxidases ChOxs are bacterial flavoproteins that catalyze the first step in the degradation of cholesterol. They contain a single molecule of FAD as the redox cofactor and .
